Current Advances in Genetic Testing for Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is one of the most common genetic disorders worldwide, and genetic testing plays a key role in its diagnosis and prevention. The last decade has seen a continuous flow of new methods for SMA genetic testing that, along with traditional approaches, have affected clinical practice patterns to some degree. Targeting different application scenarios and selecting the appropriate technique for genetic testing have become priorities for optimizing the clinical pathway for SMA. In this review, we summarize the latest technological innovations in genetic testing for SMA, including MassArray®, digital PCR (dPCR), next-generation sequencing (NGS), and third-generation sequencing (TGS). Implementation recommendations for rationally choosing different technical strategies in the tertiary prevention of SMA are also explored.


INTRODUCTION
Spinal muscular atrophy (SMA) is one of the leading genetic causes of infant and childhood mortality worldwide [1].Biallelic loss-of-function mutations in the survival of the motor neuron 1 gene (SMN1, OMIM #600354) contribute to the principal reason for SMA.The homozygous deletion of SMN1 exon 7 accounts for about 94% of SMA patients, while compound heterozygous, where a deletion on one allele and one subtle mutation on the other one is responsible for the rest cases [2].The survival of the motor neuron 2 gene (SMN2, OMIM #601627) plays a crucial role in disease phenotype modification, and the disease severity shows an inverse correlation with the copy number (CN) of SMN2 [3,4].The two SMN genes exhibit a high degree of homology, with a mere variation of 5 base pairs between them [5].
In 2001, the European Molecular Genetics Quality Network (EMQN) established practice guidelines for conducting molecular analysis in patient diagnosis, carrier detection, and prenatal diagnosis of SMA.These guidelines utilize the molecular epidemiological characterization of the disease [6] and outline the principles of various techniques, including single-strand conformation polymorphisms (SSCP), restriction enzyme digestion, linkage analysis, and competitive amplification.Meanwhile, the EMQN emphasized that *Address correspondence to this author at the United Diagnostic and Research Center for Clinical Genetics, Women and Children's Hospital, School of Medicine & School of Public Health, Xiamen University, Xiamen, Fujian 361003, P.R. China; Tel: 86-0592-7805029; E-mail: jiangyu98@xmu.edu.cn the detection performance of any method aimed at quantifying the SMN copy number should be thoroughly validated in the context of genetic testing applications for SMA.A decade later, the American College of Medical Genetics and Genomics (ACMG) proposed technical standards and guidelines for SMA genetic testing [7].In this guideline, the advantages and disadvantages of four common genetic analysis techniques, restriction fragment length polymorphism (RFLP), multiplex ligation-dependent probe amplification (MLPA), quantitative PCR (qPCR), and Sanger sequencing, are discussed in different scenarios of clinical application (Table 1).Among these methods, MLPA is universally regarded as the gold standard for diagnosis of SMA or carrier screening so far [8].

MALDI-TOF Mass Spectrometry
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been employed in the field of biochemical analysis for over two decades [43].In recent years, a MALDI-TOF MS system named MassArray ® has provided sensitive and rapid detection of single-nucleotide polymorphisms (SNPs) and has gained popularity in pharmacogenetics [44], tumor profiling [45], liquid biopsy [46], hereditary genetics [47], and methylation analysis [48].The principle of MassArray ® for genotyping assays is that the designed oligonucleotide primers would anneal directly to the target DNA in front of the SNP, followed by extension by one base in the presence of all four dideoxy nucleotides (ddNTPs).Subsequently, this base is identified by the time it takes for the extension primer mass to traverse the entire flight tube [43,49].In 2019, Lin et al. [15] reported the use of the MassArray ® technique for SMA genetic testing.In this study, primers for PCR and singlebase extension were designed to target the c.840 and c.1155 loci of the SMN gene.After successfully validating one positive case in 167 previously genotyped dried blood spot (DBS) samples with known SMN CN, three neonates with SMA were identified among 29,364 Chinese newborns.As the first report of the MALDI-TOF MS-based technique in SMA genetic testing, these results demonstrate the feasibility of its use in SMA newborn screening.However, the inability to simultaneously analyze SMN2 copy number and carrier status were major limitations of their work.Jin et al. [16] combined competitive PCR and MALDI-TOF MS (termed MS-CNV) for simultaneous quantification of the copy numbers of SMN1 and SMN2 using a 4-plex reaction system.To determine the SMN CN, a two-step normalization strategy was performed.Of the 141 cases analyzed, 24 patients, as well as 22 carriers, were successfully identified.Notably, the cut-off values for SMN CN determination were not presented in this paper; however, the authors claimed that ambiguous values of SMN2 CN were observed in a small subset of patients for MS-CNV due to inaccuracies in peak signal intensity measurement.In addition to these two reports, MALDI-TOF MS has also been employed in the study of methylation modification of SMN2 [48].Overall, SMA genetic testing based on the MALDI-TOF MS platform has not been widely used until now, and the high price of the platform instrument and the complexity of the experimental design are considered the main factors hindering its clinical application [16].

Digital PCR
Owing to the continuous reduction in equipment costs and a wide range of application scenarios, real-time quantitative PCR (qPCR) instruments have become universally used in medical laboratories worldwide.Therefore, several SMA genetic testing methods have previously been developed based on qPCR platforms [9-13, 27, 50-57].Collec-  tively, the analytical strategy to detect SMN gene copy number using qPCR can be summarized as the following two main categories: relative quantification analysis based on ΔΔCT values and the normalized ratio of product peak area or peak height (Fig. 1).Quantitative PCR-based approaches are more suitable as first-tier testing tools for patient diagnosis and newborn screening [9,10,27,51] than carrier screening and SMN2 copy number determination [54,56].The primary factor is the inherent challenge of precisely discerning various copy number combinations of SMN genes using non-absolute quantitative methods [12,27,51,56].
In recent years, digital PCR (dPCR) instruments (also known as third-generation PCR) have been rapidly developed for molecular testing.Unlike qPCR, dPCR does not require a calibration curve to assign a numerical measure of copy number.DNA molecules are randomly distributed among tens of thousands of partitions in dPCR; therefore, copy number measurements are more precise and reliable than qPCR.Current digital PCR platforms are primarily based on microfluidic or microdroplet technologies (Table 2).
Using a microdroplet-based rain dance droplet dPCR system, Zhong et al. [17] reported a pilot study using a single-tube 5-plex assay for SMA with FAM and VIC fluorophores with different fluorescence intensities to simultaneously measure the CN of SMN1/2 exon7, and genotyping an SNP of SMN1:c.815A>G.As the first report on the application of the digital PCR method in SMA genetic analysis, the authors verified only one patient and four carriers and did not present cut-off values for SMN CN.Using a similar approach, Vidal-Folch et al. [21] developed a two-tube reaction digital PCR assay on a Bio-Rad QX200 Droplet Digital PCR system for the detection of SMN1/2 copy numbers, as well as one SNP associated with the silent carrier.Using theoretical values as cut-offs for determining the SMN copy number and comparing the dPCR results of the validation set with the MLPA results, the authors obtained an accordance rate of 84.6% (14/17).In the verification sample set, digital PCR successfully identified 12 SMA-positive DNA samples and one SMA neonate from 1,530 DBS samples.In addition, the authors identified two individuals with potential silent carrier status among 125 subjects of Ashkenazi Jewish and other ancestries using a dPCR assay.Based on these results, the authors believed that they established a rapid and accurate approach to detect patients with SMA and increase the sensitivity of SMA carrier status.Park et al. [23] and Baker et al. [27] used the same platform for SMA genetic analysis using the accompanying commercial reagent for SMA genetic testing.The validation results of both studies were consistent with those of the reference method.
In 2015, Stabley et al. [18] employed a microfluidicsbased array dPCR platform to detect CN in SMN1/2 exon 7 and achieved a 100% concordance rate with the reference method for SMN1 CN in 42 validation set samples.However, they found that the concordance decreased to 80% (12/15) for samples carrying more than two SMN2 CNs in the validation set.Based on this observation, the authors suggested that dPCR could provide a more accurate measure of SMN2 CN than qPCR.Subsequently, the authors established an assay to detect SMN gene conversion or partial deletion events by increasing the detection target [25].With the improvement of the platform performance, Jiang et al. [22] developed a single-tube 4-plex reaction system to simultaneously detect SMN1/2 exon 7 and intron 1 on the QuantStudio™ Absolute Q Digital PCR System.In contrast to the approach of Vidal-Folch et al. [21], Jiang et al. established a threshold based on the results obtained from ten control samples with known SMN copy numbers.A concordance rate of 93.3% (14/15) was achieved for the verification set.Another single-tube multiplex microdroplet dPCR assay for SMA genetic testing was conducted by Tan et al. [28] using the TD-2 Droplet Digital PCR System.The authors defined the extreme values obtained from 20 replicates of four samples with varying SMN copy numbers as cut-off values, resulting in an overall agreement rate of 95.9% (291/304).Meanwhile, the authors proposed that the droplet dPCR technique requires a smaller amount of DNA and yields more precise analysis outcomes than MLPA in the quantification of SMN copy numbers because 13 DBS samples could not be accurately detected by MLPA.Similarly, Wang et al. [26] reported a method for SMN copy number detection based on a chip-in-tube digital PCR platform by establishing a threshold range using extreme values from the validation cohort in 214 samples and obtained analytical results consistent with MLPA in 12 validation samples.
In addition to the aforementioned common samples, digital PCR has been preliminarily investigated for SMA genetic testing in non-traditional sample types.Recently, Gao et al. [29] employed a microdroplet-on-chip dPCR platform to develop a novel approach for detecting male SMA carriers using semen samples, including those from silent carriers.However, the authors also noted that relying solely on dPCR results cannot distinguish the "2+0" from the "1+0" genotype.To accurately differentiate between these genotypes, combining MLPA analysis of peripheral blood DNA with digital PCR analysis of sperm cells is recommended.This presents a significant constraint in this study.Additionally, semen is not commonly used as a sample source for largescale SMA carrier screening.Thus, further evaluation of the clinical utility of this approach is required.
Over the past decade, there has been no shortage of reports detailing the implementation of digital PCR technology in the non-invasive prenatal diagnosis of monogenic genetic diseases [58][59][60][61][62][63][64].In 2020, Wei et al. [24] developed a haplotype-free dPCR assay for the non-invasive prenatal diagnosis of SMA.Of the 92.6% (25/27) samples for which classifiable results were obtained, the SMN1 copy numbers of fetal in maternal cell-free DNA were consistent with those obtained from amniotic fluid samples subjected to MLPA analysis.This report highlights the potential of dPCR for the non-invasive prenatal diagnosis of SMA; however, further studies with larger sample sizes are required before this strategy can be reliably applied in clinical settings.The MLPA results of three samples were unable to be assigned a precise copy number due to their dosage quotient falling within a grey range according to the manufacturer's instructions; b Thirteen DBS samples could not be accurately detected by MLPA due to low DNA concentration.

Next-generation Sequencing
Over the past decade, next-generation sequencing (NGS) has been widely used to diagnose genetic diseases.Recently, NGS-based expanded carrier screening (ECS) has emerged as a potent tool for preventing birth defects caused by severe autosomal recessive (AR) and X-linked conditions [65].However, the initial bioinformatics analytics model for NGS is often inadequate for analyzing CNVs in highlyhomologous sequences, such as SMN genes, and additional complementary methods, such as qPCR, are necessary for targeted SMA testing [66].In 2015, Larson et al. [30] utilized a Bayesian hierarchical model to evaluate the probability of an individual being an SMA carrier based solely on their SMN1/2 reads obtained from NGS data.This study demonstrated the initiation of SMN copy number analysis utilizing NGS data (Table 3).Using this model, the authors effectively identified four carriers out of 71 validation set samples.Subsequently, 16 individuals with a high probability of being carriers and 109 individuals with possible carri-er status were identified from the publicly available NGS dataset of 2,501 samples.In 2021, Zhao et al. [37] utilized a modified Bayesian hierarchical model for the SMA carrier screening of 10,585 diverse couples in China, obtaining an overall carrier frequency of 1.4% for the Chinese population.Subsequently, by parallel comparison, the authors proposed that NGS is relatively more reliable for SMN1 gene copy number detection because its repeatability is higher than that of qPCR and has the lowest retest rate among MLPA, qPCR, and NGS [38].
In 2017, Feng et al.
[31] developed a bioinformatics approach termed paralogous gene copy-number analysis by ratio and sum (PGCNARS) to detect SMA carriers using SMN1/2 reads at six loci of interest, achieving detection rates ranging from 90.3% to 95.0% in five ethnic groups.Based on Feng's bioinformatics strategies, Shum et al. [40] integrated an NGS assay into an existing newborn screening program and established a laboratory workflow for adding SMA to newborn screening panels.After obtaining unanimous results from 12 positive and four negative samples, the aforementioned method was utilized to screen 2,552 DBS samples; however, no positive cases were identified.
In 2020, Ceylan et al. [32] validated an SMN target sequencing assay in eight pre-characterized samples and demonstrated a 97.5% concordance rate with the expected results for 39 out of the 40 data points evaluated.Subsequently, they examined an independent cohort of 80 clinically well-characterized samples from the Turkish population to identify carriers and affected individuals with SMA and achieved a perfect correlation rate of 100%.Similarly, Huang et al. [39] employed targeted NGS of SMN genes to verify SMN1 copy numbers in 75 carriers selected from a pre-characterized cohort of 5,200 individuals using qPCR as well as ascertaining the distribution of copy numbers for the SMN2 gene among these carrier individuals simultaneously.
In 2020, Chen et al. [33] developed an NGS-based approach for distinguishing the copy number of SMN1/2 genes by analyzing the read depth and eight discerning reference genome variations.This study demonstrated a concordance rate of 99.8% for SMN1 CN and 99.7% for SMN2 CN compared to orthogonal methods, with a sensitivity of 100% for patients and 97.8% for carriers.In the same year, Liu et al. [34] presented a workflow for analyzing SMN copy numbers using uniquely mapped reads from both SMN exon 7 and the control region.The authors established cut-off values based on perfect matching results obtained from a validation cohort (n=104) and subsequently identified eight patients and 60 carriers with different SMN CN in the verification set (n=3,734).Furthermore, the authors conducted a comparative analysis between the proposed method and those developed by Larson et al. [30] and Feng et al. [31], who demonstrated that the proposed method exhibited a superior detection rate and lower incidence of false positives than the aforementioned approaches.
Tan et al. [35] provided evidence supporting the diagnostic effectiveness of incorporating SMN1 and SMN2 analyses into a comprehensive multi-gene panel designed for neuromuscular disorders.The validation assay was conducted on a cohort of 68 individuals who had been previously diagnosed with SMA by qPCR.The concordance rates for SMN1 and SMN2 CN between NGS and qPCR were 100% and 93% (63/68) within the validation cohort, respectively.Furthermore, subsequent analyses revealed that all five inaccurate results originated from qPCR.
Similar to dPCR, NGS has also been explored for noninvasive prenatal diagnosis of SMA.Chen et al. [67] utilized targeted capture sequencing, focusing on 2,011 single nucleotide polymorphisms (SNPs) within a 3-megabase interval surrounding the SMN1 gene, to conduct a pilot study on non-invasive prenatal diagnosis of SMA.This approach allowed the construction of haplotypes associated with pathogenic variants in the proband and employed a relative-haplotype-dosage (RHDO) analysis strategy.The authors successfully performed non-invasive prenatal diagnosis in five pregnant women with a family history of SMA, and their findings were consistent with those obtained through invasive prenatal diagnosis.

Third-generation Sequencing
Although dPCR and NGS-based methods have demonstrated clinical utility in SMA genetic analyses over the past few years, they still exhibit limitations in localizing small variations and detecting silent carriers.Recently, Li et al. [41] introduced a third-generation sequencing (TGS)-based approach called Comprehensive Analysis of SMA (CAS-MA), which employs long-range PCR and TGS.In this study, the sensitivity and specificity of CASMA in identifying "1+0" SMA carriers were 100% and 99.2%, respectively.CASMA is anticipated to enhance the detection rate of SMA carriers from 91% to 98% because of its ability to identify minor variants and silent carriers.However, the authors stated that the reliance of CASMA on haplotype analysis presents challenges in accurately determining copy number in instances where both SMN1 and SMN2 have the same haplotype and a read ratio of 1:1, albeit in rare occurrences.A comparable analytical approach was employed by Chen et al. [42].In this study, a high concordance rate of 99.2% (118/119) for SMN1 CN and a perfect concordance rate of 100% (116/116) for SMN2 CN were achieved using TGS.Also, this study identified two SMN1 haplotypes that demonstrated a robust association with the silent carrier phenotype in African populations.

RECOMMENDATION OF TECHNICAL STRATE-GIES FOR SPINAL MUSCULAR ATROPHY GENET-IC TESTING
Currently, the use of traditional methods, such as SSCP and RFLP, mentioned in the ACMG guidelines, has significantly declined in clinical practice.In addition to traditional methods based on qPCR or capillary electrophoresis, new SMA genetic detection approaches based on dPCR, NGS, and TGS have been developed and commercialized in recent years (Table 4).However, there are significant differences in the cost of testing, turnaround time, types of variant detection, and testing performance between these methods.Given the plethora of platforms and approaches currently available for SMA genetic testing, selecting an appropriate test strategy for various application scenarios is crucial for reducing medical expenses, shortening turnaround times, and enhancing detection rates.Based on literature reports and our clinical experience, we herein recommend a laboratory test flow for SMA genetic testing in three distinct application scenarios (Fig. 2).
The priority strategy for SMA newborn screening is to detect category 1 mutations (homozygous SMN1 exon 7 deletion) using cost-effective, high-sensitivity, and highthroughput methods [7,68].Therefore, both qPCR platform-based probe-melting curve analysis [13] and the HRM technique [9][10][11] are suitable in this scenario.A positive result from newborn screening is recommended to be confirmed using a secondary method while simultaneously acquiring SMN2 CN information [27,69].Currently, digital PCR, particularly single-tube multiplex reaction systems, is considered the most appropriate technique for the determination of SMN2 CN [22,28].The recommendation of digital PCR over MLPA, which has traditionally been considered the gold standard for SMA diagnosis, is based on existing evidence suggesting the unsatisfactory accuracy of MLPA

False positive
Neg in detecting high SMN2 copy numbers.In a retest of patients with SMA using the same MLPA assay for SMN2 CN, Schorling et al. [70] found discordant results in nine of 20 (45%) cases.This issue was also observed in our practical experience.
A comparable strategy could be applied to genetically diagnose suspected SMA patients, where dPCR can verify positive findings from qPCR or HRM analysis and provide information on SMN2 CN.If heterozygous deletion of SMN1 is detected using dPCR in suspected SMA patients, long-read sequencing methods are recommended for detecting rare variants on the other allele [41,42].Otherwise, a differential diagnosis should be conducted using NGS [35].
Owing to the severity of the disease, professional organizations recommend periconceptional carrier screening for SMA in all couples, regardless of race or ethnicity [71].For SMA carrier screening, both sensitivity and specificity are the key performance measures; therefore, methods with a high area under the curve (AUC) value from the receiver operating characteristic (ROC) curve should be considered first; second, cost, throughput, and turnaround time should be taken into account.To target a single disease for SMA, we recommend dPCR as the preferred method because of its high accuracy and low rate of false positives, whereas a positive result from NGS-based SMA carrier screening should be verified by a confirmatory test because of its relatively low sensitivity and higher false-positive rates compared to dPCR [37].

CONCLUSION
In this review, we summarize the recent technological innovations in genetic testing for SMA.Despite the strengths of high throughput, few laboratories currently use the MALDI-TOF MS platform for the clinical application of SMA genetic testing because of the high cost of instrumentation and complexity of the experimental design.Digital PCR technology, owing to its inherent feature of absolute quantification, exhibits the highest resolution for determining SMN CN among currently available technologies and does not require a complex experimental design.Consequently, it is deemed suitable as a primary technique for SMA carrier screening as well as for validating and quantifying SMN2 CN in patients with homozygous deletions.Currently, NGS technology is widely available and represents an optimal approach for implementing ECS, as well as a powerful tool for detecting minor variants and performing patient diagnoses of neuromuscular disorders, including SMA.However, given the inherent limitations of short read lengths, a complementary approach is necessary to confirm positive testing results.Compared to NGS, long-reads-based strategies have a natural advantage in the analysis of complex nucleic acid structures [72][73][74].However, its high cost and relatively low throughput limit its application in largescale population screening, making it more appropriate as a second or third-tier analysis for patients suspected of having SMA.
Although new technologies for the genetic testing of SMA are constantly emerging, it is crucial to emphasize the importance of performance validation procedures before these technologies can be implemented in clinical settings.It is worth noting that all the aforementioned novel approaches, akin to MLPA and qPCR, employ semi-quantitative methodologies to determine the copy numbers of SMN genes.Following the ACMG guidelines [7], it is imperative for laboratories conducting these tests to establish nonoverlapping validated cut-off values, ensuring accurate and reliable differentiation of SMN copy numbers of 0, 1, 2, and 3.Meanwhile, the accuracy, precision, and confidence of SMN CN measurements around these established cut-off values should be known to the laboratory.However, most of the studies reviewed herein did not provide explicit methodologies for determining the cut-off values.We recommend that laboratories develop SMN CN quantification assays following the guidelines of the Clinical and Laboratory Standards Institute [75] to establish the cut-off value using receiver operating characteristic curve analysis.Performance validation, including accuracy, precision, and limit of detection metrics, is necessary before clinical application based on a defined cut-off value.

Table 1 . Comparison of traditional SMA genetic testing methods presented in ACMG standards and guidelines [7].
Abbreviations: ACMG, American College of Medical Genetics and Genomics; RFLP, Restriction fragment length polymorphism; qPCR, Quantitative PCR; MLPA: Multiplex Ligation-dependent Probe Amplification; CN: copy number; DQ: dosage quotient; NA: not applicable; SMN1: survival of motor neuron 1 gene; SMN2: survival of motor neuron 2 gene a Only testing SMN1 copy number status; b Testing of two SNP loci, g.27134T>G and g.27706-27707delAT, which are considered associated with an increased risk of silent carriers; c See instruction protocol; d indicates minor variants.The plus sign (+) denotes applicable, more plus signs indicate more appropriate, and the minus sign (-) denotes not applicable.